ADAM (A Disintegrin And Metalloprotease) metalloproteases cleave extracellular domains of membrane-tethered proteins in a regulated, substrate-specific manner, yet without apparent preference for a cleavage sequence signature. Among other substrates, ADAM10 and ADAM17 cleave ligands for several receptor tyrosine kinase ligands, including EGF, HB-EGF, TGF-α, amphiregulin, betacellulin, epiregulin), the TNF-receptor ligand TNF-α and ephrins (Steals & Courtneidge, 2003, Genes & Dev. 17 7-30). Ephrin cleavage by ADAM10 enables cell-contact repulsion between Eph and ephrin expressing cells to proceed (Hattori et al., 2000, Science 289 1360), but how this is regulated has remained unclear.
Eph receptors and their membrane-bound ephrin ligands mediate cell positioning and pathfinding during development. Ephrin-Eph interactions on opposing cells lead to bi-directional signalling and typically induce cell-cell repulsion. Upon cell-cell contact, the interacting Ephs and ephrins form heterotetramers, which are further assembled in large signalling clusters (Himanen & Nikolov, 2003, Trends Neurosci. 26 46-51; Wimmer-Kleikamp et al., 2004, J. Cell Biol. 164 661-666).
This creates multivalent molecular tethers between opposing cell surfaces of the interacting cells that must necessarily be overcome to enable cell repulsion and signal termination.
To date two general mechanisms have been proposed that allow termination of Eph/ephrin-mediated cell contacts. EphB4-ephrinB2 interaction is thought to induce trans-endocytosis of the entire ephrin-Eph complex into either cell, in a manner dependent on the intracellular domains of both ephrin and Eph (Zimmer et al., 2003, Nat. Cell Biol. 5 869-78), and on Rac signalling (Marston et al., 2003, Nat. Cell Biol, 5 879-88).
An alternative mechanism involves ephrin cleavage by transmembrane proteases, first observed for the GPI (glycosylphosphatidylinositol)-anchored ephrin-A2, which is cleaved by the metalloprotease ADAM10. As this cleavage is essential for disrupting the Eph/ephrin cell tether, expression of an uncleavable ephrin-A2 mutant inhibits axon repulsion in neuronal cells (Hattori et al., 2000, supra). Ephrin-A2 cleavage was found to be enhanced by the presence of the EphA3 extracellular domain, and a conserved region within the Eph-binding domain of ephrins (Himanen et al., 1998, Nature 396 486-91), that appeared to promote cleavage by ADAM10, was suggested as a candidate interaction interface. The subsequently determined structures of two Eph/ephrin complexes (Himanen et al., 1998, supra; Himanen, et al., 2004, Nat. Neurosci. 7 501-509) revealed, however, that this ephrin region is involved in receptor binding and would be unavailable for ADAM interactions upon formation of an Eph/ephrin complex.
It therefore still remained unclear how ADAM proteases interact with ephrins, and how this might be regulated to ensure cleavage of only Eph-bound ephrin molecules.